Molecular pathogenicity of 1-nonadecene and l-lactic acid, unique metabolites in radicular cysts and periapical granulomas

Recently, 1-nonadecene and l-lactic acid were identified as unique metabolites in radicular cysts and periapical granuloma, respectively. However, the biological roles of these metabolites were unknown. Therefore, we aimed to investigate the inflammatory and mesenchymal-epithelial transition (MET) effects of 1-nonadecene, and the inflammatory and collagen precipitation effects of l-lactic acid on both periodontal ligament fibroblasts (PdLFs) and peripheral blood mononuclear cells (PBMCs). PdLFs and PBMCs were treated with 1-nonadecene and l-lactic acid. Cytokines’ expression was measured using quantitative real-time polymerase chain reaction (qRT-PCR). E-cadherin, N-cadherin, and macrophage polarization markers were measured using flow cytometry. The collagen, matrix metalloproteinase (MMP)-1, and released cytokines were measured using collagen assay, western blot, and Luminex assay, respectively. In PdLFs, 1-nonadecene enhances inflammation through the upregulation of some inflammatory cytokines including IL-1β, IL-6, IL-12A, monocyte chemoattractant protein (MCP)-1, and platelet-derived growth factor (PDGF) α. 1-Nonadecene also induced MET through the upregulation of E-cadherin and the downregulation of N-cadherin in PdLFs. 1-Nonadecene polarized macrophages to a pro-inflammatory phenotype and suppressed their cytokines’ release. l-lactic acid exerted a differential impact on the inflammation and proliferation markers. Intriguingly, l-lactic acid induced fibrosis-like effects by enhancing collagen synthesis, while inhibiting MMP-1 release in PdLFs. These results provide a deeper understanding of 1-nonadecene and l-lactic acid’s roles in modulating the microenvironment of the periapical area. Consequently, further clinical investigation can be employed for target therapy.

www.nature.com/scientificreports/ most predominant cells within the periapical area that play a critical role in the remodeling process and wound healing 7 . Furthermore, the periapical area was predominantly infiltrated with lymphocytes, plasma cells, and macrophages 8 . The inner layer of radicular cyst cavity is composed of stratified squamous epithelium with an outer wall of dense fibrous capsule infiltrated with chronic inflammatory cells 9 . These epithelial cells are expected to arise from the epithelial rests of Malassez 10 , while a recent study reported the possible conversion of dermal fibroblasts to epithelial cells 11 . However, the underlying mechanism is not reported. On the other hand, periapical granulomas consist of an organized profuse collagen fibers in diverse directions appearing as irregular dense connective tissue with vascular elements 12 .
PdLFs can act as immune cells and secret different cytokines 13 . The main reparative function of PdLFs is to secrete extracellular matrix components such as collagen, that builds up the periodontal ligament and its fibers 14 . Besides, macrophages are important components of the inflammatory process 15 in response to microbial infiltration in the periapical area and hence shaping up a specific periapical lesion 16 . Microbial metabolites are more predominantly contributing to such inflammatory conditions 17 .
In response to microbial entrance to the apical area, the host mounts a series of immunological and metabolic reactions that result in the destruction of periapical tissue and the development of cysts and granulomas 3 . Radicular cysts and periapical granulomas are among the most frequently occurring pathological lesions in the alveolar bone, accounting for 95% of the periapical radiolucency 18 . Interestingly, a radicular cyst may evolve from a previous periapical granuloma 19 , indicating a metabolic change in the lesion microenvironments for the induction of each lesion 3 . Thus, understanding the inflammatory and immunological roles of the major unique metabolites in the initiation of such lesions may provide potential diagnostic and therapeutic values.

Results
The effects of different concentrations of 1-nonadecene and l-lactic acid on PdLFs and PBMCs were investigated as follows:

1-Nonadecene induced MET in PdLFs.
At the gene expression level, E-cadherin was significantly upregulated at all concentrations after only 2-day treatment ( Fig. 2A and Supplementary Fig. 1), while downregulated at 1 µM and 10 µM after 6-day treatment ( Fig. 2B and Supplementary Fig. 1). Conversely, N-cadherin did not show any change after 2-day treatment ( Fig. 2C and Supplementary Fig. 1) but was significantly downregulated at all concentrations after 6-day treatment ( Fig. 2D and Supplementary Fig. 1). The expression of E-cadherin was significantly increased at both 1 µM and 10 µM after 2-and 6-day treatments (Fig. 2E,F and Supplementary  Fig. 1). On the other hand, significant decrease in N-cadherin surface protein was observed at all concentrations after 2-and 6-day treatments (Fig. 2G,H and Supplementary Fig. 1).
The release of cytokines from PBMCs was correlated to l-lactic acid's effect. The release of cytokines from PBMCs following the treatment of l-lactic acid at different concentrations was tested (Supplementary Fig. 6A-L). Compared to untreated cells, l-lactic acid did not induce any significant change in the concentrations of IL-1β, and IL-6 ( Supplementary Fig. 6A,B), all concentrations of l-lactic acid showed significant decrease in the release of IL-10, (Supplementary Fig. 6C), and only 10 µM l-lactic acid induced a significant reduction in IL-12p70 ( Supplementary Fig. 6D). Although IL-13 was not changed ( Supplementary  Fig. 6E), CXCL9 and CXCL10 showed significant reduction at all concentrations of l-lactic acid (P˂0.0001) (Supplementary Fig. 6F,G). CCL17 did not show any significant change in the treated PBMCs ( Supplementary  Fig. 6H), however, both 10 µM and 100 µM l-lactic acid significantly reduced the release of CCL22, IL-23, and TNF-α ( Supplementary Fig. 6I-K). Finally, IFN-γ was not changed at any concentration of l-lactic acid (Supplementary Fig. 6L).

Discussion
1-Nonadecene modulates a pre-invasive microenvironment that may participate in the formation of radicular cyst and related MET phenomenon. 1-Nonadecene has been identified as a microbial metabolite 5,6 , however, its biological activity is still unknown. Our previous study showed that 1-nonadecene was the highest unique metabolite identified in the radicular cysts 3 . Interestingly, further investigation confirmed that the metabolite's effect on inducing an inflammatory microenvironment is compatible with previous clinical findings 3 . IL-1β, IL-6, and IL-12A were upregulated in PdLFs and PBMCs, while IL-1α was only     20 . Further, IL-6 plays an important role in the pathogenesis of radicular cysts. IL-12A is important in CD8 + T cell clonal expansion and the generation of memory CD8 + T cells 21 . Autophagy pathway was the highest enriched metabolic pathway in radicular cysts 3 and was found as a marker of cytotoxic CD8 + T cells 22 . 1-Nonadecene upregulated MMP-1 gene expression in PdLFs and PBMCs. MMP-1 contributes to bone resorption and cyst expansion. Epithelial proliferation was noticed to be high in radicular cysts 23 . For insistence, 1-nonadecene caused the upregulation of VEGFα and PDGFα. VEGFα has been reported to be an important factor in angiogenesis, cell proliferation and differentiation, and microvascular permeability. This can lead to extravasation of plasma proteins, fluid accumulation, and edema in radicular cysts 24 . PDGFα is necessary for the stabilization and maturation of the newly formed blood vessels in addition to endothelial cell differentiation 25 (Fig. 6A). The data were analyzed using one-way ANOVA and Dunnett's multiple comparisons test. P-value < 0.05 was considered significant.  10 . Here, we confirmed that this pathogenesis is correlated to MET www.nature.com/scientificreports/ phenomenon by measuring the expression of E-cadherin and N-cadherin, consistent with previous report 26 . Epithelial cells are predominant in the lining of radicular cysts 9 and can be derived from mesenchymal fibroblast cells 11 . Both N-cadherin and E-cadherin are affected by 1-nonadecene treatment, concluding that the same pathway is involved at both the protein and gene expression levels (Fig. 2). Previous studies showed that runt-related transcription factor (RUNX)-2 is highly expressed in radicular cyst lining 27 and it simultaneously upregulates E-cadherin and downregulates N-cadherin, while TGFβ1 reverses this expression 28 . TGFβ1 was downregulated in PdLFs and PBMCs after treatment with 1-nonadecene (Fig. 1M,Y), which was clinically confirmed by the significantly low level in radicular cysts 29 . These findings reveal that 1-nonadecene stimulates the expression of RUNX2 that mediates the MET and at the same time downregulates TGFβ1 for further MET (Fig. 6A).
The transcriptomic results revealed a proinflammatory effect of 1-nonadecene on PBMCs (Fig. 1S-AA). Furthermore, 1-nonadecene upregulates the expression of CD14 high /CD16 low , HLA-DR/CD86, CD163, and CD206 in M1 macrophages, indicating a high polarization towards M1 and to be activated for phagocytosis and healing process at the same time. This result indicates the possible participation of reactive oxygen species-producing M1 in bone resorption and cystic expansion 30 . In comparison to periapical granulomas, HLA-DR was significantly upregulated in the radicular cysts 31 , while CD86 expression has not been identified previously in radicular cysts and needs further investigation. The activated pathway induced by 1-nonadecene still needs to be further investigated in the future.
Collectively, 1-nonadecene modulates a microenvironment that may be involved in the induction of the radicular cyst and the transformation of PdLFs to epithelial cells, thereby forming the cyst's lining. l-lactic acid modulates a pre-invasive microenvironment that may enhance the formation of periapical granuloma and related fibrosis. Although l-lactic acid was observed to cause facial granulomas after cosmetic poly-l-lactic acid injection 4 , no study investigated its pathogenic immunological role in the formation of granulomatous lesions in other body parts. In this study, we found that PdLFs and PBMCs treated with l-lactic acid exert differential anti-inflammatory and proliferative effects. In consistence, microbial lactic acid downregulates IL-1β and IL-6 in epithelial cells 32 . Although the direct effect of l-lactic acid on IL-1α in PdLFs or PBMCs has not been studied before, we observed an upregulation of IL-1α in the late treatment of PdLFs (6 days), but downregulation in PBMCs. MCP-1 showed both patterns of upregulation and downregulation at different concentrations, time points 33 , and cell types 34 . Additionally, some studies showed a proliferative effect of lactic acid through the elevated levels of VEGFα 35 and PDGFα 36 in macrophages, while in our study, these markers were downregulated in PBMCs. On the other hand, we observed both upregulation and downregulation of VEGFα and PDGFα in PdLFs. These results were expected since we have used different concentrations and times for the treatment (Fig. 6B).
Another promising result regarding the metabolic shift induced by l-lactic acid is the increase in collagen precipitation, which is accompanied by a simultaneous decrease in MMP-1. A high level of MMP-1 was found in aerobic glycolysis, while oxidative phosphorylation enhances collagen synthesis 37 . Hence, we concluded that l-lactic acid may induce oxidative phosphorylation in periapical granulomas represented by high collagen precipitation and low ability to degrade this excess in the collagen matrix (Fig. 6B).
The effect of l-lactic acid on PBMCs revealed that it did not induce direct M2 polarization without concomitant M1 polarization. Previously, it has been shown that lactic acid can induce M2 polarization in an environment with previous inflammation 38 . In our study, there was no previous inflammatory stimulation of PBMCs; therefore, we concluded that direct l-lactic acid treatment induces M1 polarization with concomitant activation markers, HLA-DR, CD86, and CD163 for both M1 and M2 macrophages. This means that there is an activation of both classical and non-classical macrophages, but whether this activation is sufficient to release their respective cytokines is not known. Therefore, we measured the concentration of the cytokines in PBMCs' supernatants. The anti-inflammatory cytokines IL-10 and CCL22 were found to be downregulated in PBMCs. The anti-inflammatory effect of l-lactic acid is represented by inhibiting the release of IL-12 and TNF-α from PBMCs, consistent with other studies for this inhibition from dendritic cells and macrophages 38 . l-lactic acid inhibits the release of CXCL9 and CXCL10 in PBMCs. Ultimately, this inhibition mediates immunosuppression in periapical granulomas. l-lactic acid polarizes CD4 + T cells to Th-17 T cells 39 , but here we did not find that l-lactic acid induces the release of IL-23 from PBMCs to induce Th-17 differentiation. Overall, these results indicate that l-lactic acid alone inhibits the release of both proinflammatory and anti-inflammatory cytokines from PBMCs.
In summary, we demonstrated that a dominant unique metabolite could modulate a pre-invasive microenvironment that may participate in the initiation and pathogenesis of periapical lesions. 1-Nonadecene enhanced MET through the upregulation of E-cadherin and downregulation of both N-cadherin and TGFβ1. l-lactic acid increased the collagen synthesis and decreased its degradation by inhibiting MMP-1. Taken together, understanding the metabolomic consequences can dismantle disease's pathogenicity, map the significance of microbial metabolites in modulation of a microenvironment, and provide a suggested therapeutic target following further clinical investigation.
The cells were harvested and fixed in stain wash buffer (SWB) (1% sodium azide, 2% FBS in PBS) and the cells were then treated with rabbit anti-human E-cadherin Alexa 647 (Cat# 9835, RRID:AB_10828228, Cell Signaling Technology, USA) or rabbit anti-human N-cadherin (Cat# ab245117, RRID:AB_2910595, Abcam, USA) according to the manufacturer's instructions. Secondary antibody goat anti rabbit IgG Alexa 488 was used for N-cadherin (Cat# ab150077, RRID:AB_2630356, Abcam, USA). Corresponding isotype controls were used to compare the expression of the markers.
Collagen assay. Soluble collagen assay kit (ab242291, Abcam, UK) was used to detect the collagen formation in 1 × 10 6 PdLFs treated with 1 µM, 10 µM, or 100 µM l-lactic acid in comparison to 1 µM PMA for 2 and 6 days. The experiment was conducted in triplicates, and the manufacturer's instructions were followed.
Western blot assay. To investigate the protein expression of MMP-1 in PdLFs, the cells were treated with